The proximal tubule is the main target for nephrotoxic substances due to its specific properties, including efficient drug transport and biotransformation potential. The availability of a pure population of proximal tubule cells (PT cells) as a model to study a range of biological, pharmacological, and toxicological parameters is, therefore, of great value. A two-step PT cell-isolation procedure, based on density-gradient centrifugation, is described; this procedure can easily be introduced into each laboratory setting. The procedure routinely yields a highly pure PT cell population, comprising 20-40 × 10(6) cells, which can be used for preparation of subcellular fractions or brought into primary culture.